Alginate is a linear polysaccharide composed of ␤-D-mannuronate and the C 5 epimer ␣-L-guluronate, arranged in three different ways: poly-␤-D-mannuronate (polyM), poly-␣-L-guluronate (polyG), and heteropolymeric (polyMG) region in which there is a random arrangement of the monomers (4). Alginate produced by brown seaweeds is not acetylated and is widely used in food and pharmaceutical industries because of the ability of the polymer to chelate metal ions and form a highly viscous solution (18). The acetylated form of alginate is synthesized by certain bacteria, such as mucoid cells of Pseudomonas aeruginosa and Azotobacter vinelandii. P. aeruginosa causes serious chronic pulmonary infections in the lungs of patients with cystic fibrosis (CF) (2), and alginate produced by the bacterial cells seems to play a crucial role in the adherence of the bacterium to target cells (21). The alginate functions as a biofilm and decreases the effect of antimicrobial agents by repressing the penetration of the agent into the biofilm, thus making it difficult to treat biofilm-dependent bacterial infectious diseases (5).A bacterium, Sphingomonas sp. strain A1, incorporates alginate into the cells through a pit formed on the cell surface (8), and an ABC (ATP-binding cassette) transporter specific to the macromolecule is responsible for transport of alginate (16) (Fig. 1). The incorporated alginate is depolymerized by three types of cytoplasmic alginate-depolymerizing enzymes (alginate lyases A1-I [66 kDa], A1-II [25 kDa], and A1-III [40 kDa]) formed from a common precursor protein through the consecutive processes of posttranslational modification (Fig. 1) (17). A1-I is autoprocessed to give rise A1-II and A1-III (9), and these alginate lyases cleave glycosidic bonds endolytically in the alginate molecule by ␤-elimination reaction. Briefly, A1-I is active on acetylated and nonacetylated alginates. A1-II prefers polyG and nonacetylated alginate produced by brown seaweeds. A1-III efficiently liquefies polyM and acetylated alginates produced by mucoid cells of P. aeruginosa derived from the lungs of CF patients (17, 32); this property may be useful as a clinical agent for the therapy of CF and other infectious diseases caused by P. aeruginosa.Three alginate lyases (A1-I, A1-II, and A1-III) can produce di-and trisaccharides from alginate as major final products (6, 32), thus implying that the cells of Sphingomonas sp. strain A1 have an additional enzyme responsible for the degradation of alginate oligosaccharides to the constituent monosaccharides. To construct the complete metabolic pathway for alginate in Sphingomonas sp. strain A1, we have purified and characterized the enzyme (oligoalginate lyase) catalyzing the degradation of alginate oligosaccharides and determined its primary structure by cloning the gene. MATERIALS AND METHODSMaterials. Sodium alginate (average molecular weight, 25,700; polymerization degree, more than 100, viscosity, 1,000 cp) from Eisenia bicyclis and DEAEcellulose were purchased from Nacalai Tesque Co. Ltd.,...

A gram-negative bacterium, Sphingomonas sp. strain A1, isolated as a producer of alginate lyase, has a characteristic cell envelope structure and forms a mouth-like pit on its surface. The pit is produced only when the cells have to incorporate and assimilate alginate. An alginate uptake-deficient mutant was derived from cells of strain A1. One open reading frame, algS (1,089 bp), exhibiting homology to the bacterial ATP-binding domain of an ABC transporter, was cloned as a fragment complementing the mutation. algS was followed by two open reading frames, algM1 (972 bp) and algM2 (879 bp), which exhibit homology with the transmembrane permeases of ABC transporters. Disruption of algS of strain A1 resulted in the failure to incorporate alginate and to form a pit. Hexahistidine-tagged AlgS protein (AlgS His6 ) overexpressed in Escherichia coli and purified by Ni 2؉ affinity column chromatography showed ATPase activity. Based on these results, we propose the occurrence of a novel pit-dependent ABC transporter system that allows the uptake of macromolecules.Alginate is a heteropolysaccharide consisting of ␣-L-mannuronate and its 5Ј-epimer, ␣-L-guluronate, and is produced by brown seaweeds and certain bacteria. Other than its utilization in the medical, chemical, and food areas, this biopolymer has been widely associated with chronic Pseudomonas aeruginosa infection in the lungs of cystic fibrosis patients (5). A bacterium, Sphingomonas sp. strain A1, was isolated from soil as a potent producer of alginate lyase catalyzing the depolymerization of alginate (24). The cells of strain A1 are covered by many large plaits (Fig. 1a), and a mouth-like pit (0.02 to 0.1 m in diameter) is formed on their surface when they are grown in a medium containing alginate as the sole carbon source (10, 11) ( Fig. 1b and c). The pit formed in the presence of alginate disappears when the cells are transferred to a medium without alginate (10, 11). In the presence of alginate, a specific region of the cell surface and its neighborhood is intensely stained with an agent that interacts with mucopolysaccarides (alginate), and thin sections of cells show an irregular site where the cell membrane sinks deeply into the cytosol (10, 11).For the utilization of polysaccharides and other macromolecules, microbes usually degrade them by means of extracellular enzymes and then take the depolymerized low-molecular-weight (LMW) products up through their membranes. However, alginate lyase is exclusively localized in the cytoplasm, and the periplasmic and membrane fractions contain no alginate lyase activity (25). Furthermore, no extracellular alginate-depolymerizing activities have been detected in concentrated culture fluid of strain A1 (24, 25), when assayed by measuring the changes in viscosity and absorbance at 235 nm (for alginate lyase) or analysis of the depolymerization profile by thin-layer chromatography (data not shown). The amino acid sequence of alginate lyase purified from strain A1 was 53 amino acids shorter in the N-terminal region than t...

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A Gram-negative bacterium, Sphingomonas sp. A1, has a macromolecule (alginate) import system consisting of a pit on the cell surface and an alginate-specific ATP-binding cassette importer in the inner membrane. Transport of alginate from the pit to the ABC importer is probably mediated by two periplasmic binding protein homologues (AlgQ1 and AlgQ2). Here we describe characteristics of binding of AlgQ1 and AlgQ2 to alginate and its oligosaccharides through surface plasmon resonance biosensor analysis, UV absorption difference spectroscopy, and X-ray crystallography. Both AlgQ1 and AlgQ2 were inducibly expressed in the periplasm of alginate-grown cells of strain A1. Biosensor analysis indicated that both proteins specifically bind alginate with a high degree of polymerization (>100) and that dissociation constants for alginate with an average molecular mass of 26 kDa are 2.3 x 10(-)(7) M for AlgQ1 and 1.5 x 10(-)(7) M for AlgQ2. An in vitro ATPase assay using the membrane complex, including the alginate ABC importer, suggested that both alginate-bound forms of AlgQ1 and AlgQ2 are closely associated with the importer. X-ray crystallography showed that AlgQ1 consisted of two domains separated by a deep cleft that binds alginate oligosaccharides through a conformational change in the two domains. These results directly show that alginate-binding proteins play an important role in the efficient transport of alginate macromolecules with different degrees of polymerization in the periplasm.

Sphingomonas sp. A1 possesses a high molecular weight (HMW) alginate uptake system composed of a novel pit formed on the cell surface and a pit-dependent ATP-binding cassette (ABC) transporter in the inner membrane. The transportation of HMW alginate from the pit to the ABC transporter is mediated by the periplasmic HMW alginate-binding proteins AlgQ1 and AlgQ2. We determined the crystal structure of AlgQ2 complexed with an alginate tetrasaccharide using an alginate-free (apo) form as a search model and refined it at 1.6-Å resolution. One tetrasaccharide was found between the N and C-terminal domains, which are connected by three extended hinge loops. The tetrasaccharide complex took on a closed domain form, in contrast to the open domain form of the apo form. The tetrasaccharide was bound in the cleft between the domains through van der Waals interactions and the formation of hydrogen bonds. Among the four sugar residues, the nonreducing end residue was located at the bottom of the cleft and exhibited the largest number of interactions with the surrounding amino acid residues, suggesting that AlgQ2 mainly recognizes and binds to the nonreducing part of a HMW alginate and delivers the polymer to the ABC transporter through conformational changes (open and closed forms) of the two domains.

Wationai fristitute ofAgrobioiogicalResources, 71yukub ¢ Ibaraki305-085a

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Sphingomonas sp. A1 possesses specialized membrane structures termed 'superchannels' that enable the direct incorporation of macromolecules into the cell. We have engineered two related sphingomonads, the dioxin-degrading S. wittichii RW1 and the polypropylene glycol-degrading S. subarctica IFO 16058(T), to incorporate this superchannel into their cell membranes. In both cases the bioremediation capability of the organisms was substantially increased pointing at the potential of this approach as a general strategy to improve bacterial degradation of hazardous compounds in the environment.